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  • br Longer telomeres have been observed


    Longer telomeres have been observed in 14% of colon cancers
    compared to matched noncancerous mucosa, and longer telomeres were also associated with a poor prognosis in patients with colon cancers [13]. Telomerase, responsible for the synthesis of telomeres, is also reported to be expressed abundantly in highly proliferative cells, such as germ Etoposide and stem cells, and in the cells of about 85% of cancers, while its activity is nearly undetectable in most somatic cells [14,15]. As the main component of telomerase [16,17], hTERT (human telomerase reverse transcriptase) was revealed to be deregulated and largely amplified in most cancers, leading to aberrant cell proliferation, metastasis, stemness maintenance and immortalization [18–21]. Thus, strictly controlling the level of hTERT represents an efficient strategy in limiting the traits of CSCs, thereby limiting cancer progression. It is suggested that hTERT expression is tightly regulated at the transcrip-tional level by many different transcriptional factors [22–26]. However, little is known about the precise molecular regulation of hTERT in CRC
    Correspondence to: W. Guo, Institute of Cancer Stem Cell, Dalian Medical University, Dalian 116044, China.
    Corresponding authors at: Sun Yat-sen University Cancer Center, Guangzhou 510060, China.
    development, especially in its CSC sculpture progression.
    Cleavage and polyadenylation specific factor 4 (CPSF4), alter-natively known as NAR, NEB1 or CPSF30, is a member of the CPSF complex that contains six subunits: CPSF160, Wdr33, CPSF100, CPSF73, Fip1, and CPSF4 [27,28]. CPSF4 plays a key role in pre-mRNA 3′ end processing, recognizing the AAUAAA signal sequence and in-teracting with poly(A) polymerase and other factors to bring about cleavage and poly(A) addition. In other words, it is evidenced that CPSF4 participates in the maturation of mRNA [29–32]. Beyond this, CPSF complex, including CPSF4, has also been reported to interact with the tumor suppressor Cdc73 to facilitate its regulation on the down-stream target gene [33]. However, the precise role and molecular me-chanisms of CPSF complex in tumorigenesis and development are still poorly understood. Our previous work reported that CPSF4 played a pivotal role in lung cancer progression by targeting hTERT [34]. Nevertheless, it is unclear whether it plays the same role in other cancer types, including CRC, and if so, whether the realization of such function still relies on its regulation on hTERT? All these questions deserve to be better explored.
    In this study, we investigated the roles and the underlying me-chanisms of CPSF4 in colorectal cancer. Knockdown of CPSF4 sup-pressed tumor progression in vitro, represented by the reduced cell proliferation, metastasis, stemness and the increased sensibility of chemotherapeutic drug in colon cancer cells. While its overexpression caused completely reverse effects in vitro and in vivo. Furthermore, we showed the recruitment of CPSF4 to hTERT promoter (−321 to −234 locus) under the cooperation of NF-kB1, resulting in activation of hTERT gene transcription. hTERT knockdown antagonized CPSF4 overexpression-mediated oncogenic function. Of note, CPSF4 was po-sitively correlated with hTERT in colon cancer samples from patients and their simultaneously high expression predicted poor outcome. All these findings proved CPSF4 as a novel pro-tumorigenic factor in col-orectal cancer progression, and also define CPSF4-hTERT axis as a po-tential target for therapeutic intervention in colorectal cancer devel-opment.
    2. Materials and methods
    2.1. Cell lines and cell culture
    Human colon cancer cell lines RKO,SW620,LOVO and DLD1 were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in RPMI-1640 medium (Gibco) or DMEM (Gibco) supplemented with 10% fetal bovine serum (Gibco, Waltham, MA).All the cells were maintained in an environment with a humidified incubator with 5% CO2 at 37 °C.